Abstract

Purpose: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the bronchoalveolar lavage (BAL) samples of lung cancer patients.

Experimental Design: We examined the tumor and the matched BAL DNA for aberrant methylation of eight gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-β2, and ARF) from 31 patients with primary lung tumors by quantitative fluorogenic real-time PCR. BAL from 10 age-matched noncancer patients was used as a control.

Conclusion: Our findings suggest that promoter hypermethylation in BAL can be detected in the majority of lung cancer patients. This approach needs to be evaluated in large early detection and surveillance studies of lung cancer.

Introduction

Lung cancer is the leading cause of cancer-related death in the United States and other developed countries (1)
. In 2001, an estimated 169,500 new cases of primary lung cancer were diagnosed, and an estimated 157,400 people died of this disease in the United States (1)
. The survival rates for lung cancers have changed little over the past two decades. A major factor in the high mortality of lung cancer patients is the presence of metastatic tumors in approximately two-thirds of patients at the time of diagnosis (2)
.

Detection of lung cancer at earlier stages could potentially increase survival rates by 10- to 50-fold (2)
. Lung cancer screening by chest X-ray and sputum cytology have proven ineffective in increasing patient survival (3, 4)
, leading to the search for more sensitive and specific tests. One promising approach is the identification of lung cancer-specific biomarkers and noninvasive methods for the detection of these biomarkers at an early stage.

Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor or other cancer-associated genes in many kinds of human cancer (5, 6, 7, 8, 9)
. Thus far, genes such as APC, CDKN2A, CDH1, RAR-β2, and RASSF1A have been found to harbor hypermethylated promoters in over 30% of lung tumors (10, 11)
. The development of the real-time methylation-specific PCR, which is more sensitive than conventional methylation-specific polymerase chain reaction (MSP) by 10-fold, has simplified the study of the genes inactivated by promoter hypermethylation in human cancer and has the advantage of increasing specificity attributable to the use of an internally binding fluorogenic hybridization probe for each gene (12, 13)
. Recent publications have demonstrated the presence of promoter hypermethylation of various genes in bodily fluids, including bronchoalveolar lavage (BAL) DNA of lung cancer patients (14, 15, 16, 17, 18)
.

In the present study, we analyzed the promoter hypermethylation pattern of eight key genes in the primary tumor DNA of 31 lung primary tumors. The methylation pattern found in the primary tumors was compared with the methylation pattern in matched BAL fluid DNA. Approximately two-thirds of the patients with methylated tumors were found to display identical epigenetic changes in the paired BAL fluid.

Materials and Methods

Sample Collection and DNA Preparation.

We evaluated tissue samples of 31 patients with lung cancer, who underwent curative surgery between 1995 and 1999 at the Johns Hopkins University, School of Medicine. Detailed information on these patients is summarized in Table 1⇓
. Obtained tissue specimens were immediately snap frozen in liquid nitrogen and stored at −80°C. H&E-stained sections were histologically examined every 20 sections for the presence or absence of tumor cells, as well as for tumor density. Only sections that showed >70% of tumor cells were used for DNA extraction. Under the proper consent, BAL fluid was collected during flexible bronchoscopy in the operating room for the patient undergoing resection of non-small cell lung carcinoma before thoracotomy. BAL fluids from the 10 age-matched individuals with nonmalignant lung diseases were also collected during bronchoscopy under standard procedures. Briefly, after the initial examinations were performed with bronchoscopy, four 30-ml (6 teaspoons) washes of germ-free i.v. salt solution were sequentially installed and retrieved by gentle suction, pooled, and placed on ice and were then transported to the laboratory and centrifuged at 1800 × g for 10 min at 4°C. The cell pellet was then collected and stored at −80°C. DNA isolation was performed as described previously (19)
.

Bisulfite Treatment.

DNA from tumor and BAL fluid was subjected to bisulfite treatment, as described previously (20)
. Briefly, 2 μg of genomic DNA was denatured in 0.2 m NaOH for 20 min at 50°C. The denatured DNA was diluted in 500 μl of freshly prepared solution of 10 mm hydroquinone and 3 m sodium bisulfite and incubated for 3 h at 70°C. After incubation, the DNA sample was desalted through a column (Wizard DNA Clean-Up System, Promega), treated with 0.3 m NaOH for 10 min at room temperature, and precipitated with ethanol. The bisulfite-modified genomic DNA was resuspended in 120 μl of H2O and stored at −80°C.

Methylation Analysis.

Templates were amplified by a fluorescence-based real-time PCR (Taqman) as described previously (21)
. In brief, primers and probes were designed to specifically amplify the bisulfite-converted promoter of the gene of interest. The ratios between the values of the gene of interest and the internal reference gene, β-actin, obtained by Taqman analysis were used as a measure for representing the relative level of methylation in the particular sample. Fluorogenic PCRs were carried out in a reaction volume of 25 μl consisting of 600 nm each primer, 200 nm probe, 5 units of Taq polymerase, 200 μm each of dATP, dCTP, and dGTP; 400 μm dTTP; and 5.5 mm MgCl2. Three microliters of treated DNA solution were used in each real-time MSP reaction. Amplifications were carried out in 384-well plates in a 7700 Sequence detector (Perkin-Elmer Applied Biosystems). Each plate consisted of patient samples and multiple water blanks, as well as positive and negative controls. Leukocytes from a healthy individual were methylated in vitro with excess SssI methyltransferase (New England Biolabs Inc., Beverly, MA) to generate completely methylated DNA, and serial dilutions of this DNA were used for constructing the calibration curves on each plate.

Statistical Analysis.

All statistical tests were performed using Excel software (Microsoft).

Representative examples of methylation levels of APC, MGMT, RASSF1A, and CDH1 in lung cancer tissue matching bronchoalveolar lavage (BAL) samples from lung cancer patients and BAL from noncancer patients. Calculation of the gene of interest:β-actin ratio was based on the fluorescence emission intensity values for both the gene of interest and β-actin obtained by quantitative real-time PCR analysis. The relative amount of methylated promoter DNA is much higher in tumor tissue than the corresponding BAL sample. Zero values are plotted on the X-ordinate for simplicity, although they do not exist on a log scale.

Frequency of Methylation in Primary Lung Tumor.

Aberrant promoter hypermethylation of at least one of the genes investigated was detected in all 31 lung primary tumors (100%; Table 2⇓
; Fig. 2⇓
). The median methylation values (gene/β-actin X 100) for each gene in tumor and BAL DNA are shown in Table 2⇓
. The frequency of aberrant methylation of each gene is listed in Table 2⇓
. In the tumor samples, frequent methylation was detected in CDH1 (87%), APC (55%), RASSF1A (45%), and MGMT (39%). Methylation of p16 was detected in 23% of cases. Methylation of GSTP1 (10%) and RAR-β2 (10%) were much less common. Methylation of the ARF promoter DNA was not detected in any of the 31 cases.

Aberrant methylation in primary lung carcinoma had no correlation with patient demographic data, including age and gender, histological subtype, and staging of the tumor (data not shown).

Epigenetic Alterations in BAL DNA from Lung Cancer Patients.

The matching 31 BAL samples from these lung cancer patients were then tested for epigenetic alterations. Overall, 21 of 31 (68%) patients were methylation positive for at least one of the eight genes tested (Table 2⇓
; Fig. 2⇓
). BAL DNA from 0 of the 10 controls with non-neoplastic lung disease displayed promoter hypermethylation at five genes examined. CDH1, RASSF1A, and APC showed very low levels of methylation in three control BAL samples; however, the median methylation levels in these control samples for CDH1, APC, and RASSF1A were 0 in each gene and the range was 0.000–0.022, 0.147–1.760, and 0.000–0.048, respectively. For these three genes, we set the highest methylation level in normals as the cutoff value to determine methylation-positive samples for lung cancer tissue and BAL fluid. With this cutoff, the frequency of aberrant promoter methylation detected in matched BAL fluid for each marker was 48% (13 of 27) for CDH1, 58% (7 of 12) for MGMT, 29% (4 of 14) for RASSF1A, 29% (5 of 17) for APC, 33% (1 of 3) for GSTP1, 14% (1 of 7) for p16, and 0% (0 of 3) for RAR-β2 (Table 2)⇓
. The identical methylation pattern was found between primary tumor and matched BAL samples as shown in Fig. 2⇓
. As seen in tumor samples, the median methylation level for APC was the highest (median, 12.83; range, 0.000–23.282) in BAL samples (Fig. 1)⇓
. Overall, we found no correlation between the methylation index (MI = total number of genes methylated/total number of genes analyzed) and any of the clinicopathological characteristics, i.e., tumor type, grade and stage in BAL samples (data not shown).

Discussion

In this study, all 31 of primary lung tumors harbored CpG island hypermethylation of at least one of eight cancer-related genes. Three commonly methylated genes (CDH1, APC, and RASSF1A) accounted for 80% of our positive cases. Sixty-eight percent of patients with aberrant methylation in primary tumors also exhibited hypermethylation in BAL DNA. It is noteworthy that detection of promoter methylation in BAL is a specific event; i.e., (a) aberrant methylation was not detected in any of the 10 control BAL samples with the exception of negligible levels in three genes for which a cutoff was developed; (b) the identical methylation profiles were found in the corresponding tumor; and (c) aberrant methylation was not detected in the BAL of lung cancer patients without methylation in the corresponding tumor.

The development of real-time PCR has simplified the study of genes inactivated by promoter hypermethylation in human cancer. It is a highly sensitive assay that is capable of detecting methylated alleles in the presence of a 1000-fold excess of unmethylated alleles. However, it is more stringent and maybe more specific because in addition to the two PCR primers, the fluorescent-labeled hybridization probe has to anneal correctly between the two primers. The quantitative MSP is often more sensitive than conventional MSP but varies based on the promoter, primers, and condition. In a previous study, based on conventional MSP, we detected methylated p16 alleles in the BAL fluid from 63% of the patients with a methylated primary tumor (18)
. In the present study, p16 was methylated in only 14% of BAL samples with a methylated primary tumor. The reason for this discrepancy may be attributable to primer design, but it should be noted that the sample size was different in both studies, and tumor stages and grade also differed.

Several studies using different approaches have demonstrated promoter hypermethylation of CDH1 (40–55%), APC (46–96%), RASSF1A (30–40%), p16 (25%–41%), MGMT (16–27%), RAR-β (40–43%), GSTP1 (7–12%), and ARF (6–8%) in lung cancer (11, 22)
. We observed a higher frequency of methylation for CDH1 (87%), RASSF1A (45%), and MGMT (39%) in primary lung tumors. Again, these results may reflect a different sensitivity for our assay. Consistent with our findings, a recent study demonstrated MGMT promoter methylation in 38% of their cases that were examined by quantitative MSP (23)
. Although, methylation of p16, GSTP1, ARF, and APC were similar to previous reports (11)
, we found methylation for RAR-β2 in 10% of primary tumors in contrast to a higher frequency in another study (24)
. We do not have a clear explanation for this discrepancy, but assay sensitivity and small sample size may be the causes.

To our knowledge, there have been only two studies (18, 25)
using limited number of methylation markers for the detection of lung cancer using BAL samples. Thus, our methylation assay using eight different genes in the same BAL DNA confirms and extends previous observations. The specificity of four genes (MGMT, RAR-β2, GSTP1, and p16) was 100% with undetectable methylation levels in all normal samples. The detection of tumor molecular signatures in body fluids has implications for the identification of high-risk subjects, patients with preinvasive or early-stage lesions, and for monitoring residual disease. Molecular approaches characterized by a high specificity have variable sensitivity, perhaps because of the presence of only minimal tumor DNA in BAL or because of a high level of contamination with normal DNA. Several approaches to improve assay sensitivity have been applied to tumor tissue, plasma, sputum, stool, bronchoalveolar lavage, and bronchial brushings. Sensitivity was improved over conventional MSP (20)
by performing a semi-nested MSP after a DNA preamplification step (26)
or a nested two-stage PCR (17)
. We can improve the sensitivity of quantitative MSP in BAL fluid by the following: (a) isolating aberrant cells from BAL before DNA extraction; (b) adding nested or seminested PCR before quantitative MSP; and (c) increasing the number of lung cancer-specific markers. More sensitive assays, however, generally result in imperfect specificity and must be validated in clinical samples.

Exfoliative material (present in sputum, bronchoalveolar lavage, and bronchial brushings) offers diagnostic access, but the sensitivity of current cytological tests is low. Diagnostic tools that would provide high specificity and sensitivity would clearly be of enormous benefit to patients, particularly if the specimens could be obtained by noninvasive means. To this end, the detection of aberrant methylation in sputum in addition to BAL DNA may offer a promising approach for the noninvasive diagnosis of lung cancer. As seen in saliva for the detection of head and neck cancer (27)
, these approaches are highly specific and correlate with tumor methylation status. Apart from lung cancer detection, it would be interesting to see whether the detection of aberrant methylation in the sputum or BAL can be used in the monitoring of disease progress after curative surgery. If methylated DNA disappears shortly in these samples after curative surgery, the reappearance of these markers may suggest recurrence of disease that may require more intensive screening and aggressive treatment. Additional studies are necessary to elucidate the role of detecting aberrant methylation in such samples as a tool for early detection and surveillance of lung cancer.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: O. Topaloglu and M. Hoque contributed equally to this work. Under a licensing agreement between Oncogenome Sciences, SA and the Johns Hopkins University, D. Sidransky is entitled to a share of royalty received by the University upon sales of products described in this article. D. Sidransky owns Oncogenome Sciences, SA stock, which is subject to certain restrictions under University policy. D. Sidransky is a paid consultant to Oncogenome Sciences, SA and is a paid member of the company’s Scientific Advisory Board. The Johns Hopkins University in accordance with its conflict of interest policies is managing the terms of this agreement.